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Intensifying global change is propelling many ecosystems toward irreversible transformations. Natural resource managers face the complex task of conserving these important resources under unprecedented conditions and expanding uncertainty. As once familiar ecological conditions disappear, traditional management approaches that assume the future will reflect the past are becoming increasingly untenable. In the present article, we place adaptive management within the resist–accept–direct (RAD) framework to assist informed risk taking for transforming ecosystems. This approach empowers managers to use familiar techniques associated with adaptive management in the unfamiliar territory of ecosystem transformation. By providing a common lexicon, it gives decision makers agency to revisit objectives, consider new system trajectories, and discuss RAD strategies in relation to current system state and direction of change. Operationalizing RAD adaptive management requires periodic review and update of management actions and objectives; monitoring, experimentation, and pilot studies; and bet hedging to better identify and tolerate associated risks.

The Greater Adjutant Stork (Leptoptilos dubius), locally known as Hargila (the bone swallower) is an endangered bird with an estimated global population of less than 1200. Habitat loss, poisoning, and poaching have caused large declines in populations of this stork in South Asia, with the Brahmaputra valley in Assam in northeastern India now the last stronghold for the species. The stork nests colonially in privately owned trees within thickly populated villages. Tree owners would cut down trees to prevent rotten food and excreta of this carnivorous bird from falling into their backyards. A change in attitudes of the nest-tree owners towards keeping their trees and towards Greater Adjutants has been the key to stork conservation. A conservation project involving community development, education and outreach, interlinking storks with local traditions and cultures, and capacity building of local communities was initiated in 2007. A rural women's conservation group named the Hargila Army was instituted and strong feelings of pride and ownership for the storks by the villagers have been generated. Cash incentives for nest protection were deliberately avoided, with schemes that indirectly contribute to the livelihoods of nest-tree owners and other villagers introduced instead. The success of the conservation program is shown by the increase in the number of nesting colonies in the village area of Dadara, Pachariya, and Singimari in Kamrup District in Assam from 28 nests in 2007–08 to 208 nests in the 2019–20 breeding season, making this the largest breeding colony of Greater Adjutant Storks in the world.

The Kenai National Wildlife Refuge has been given a broad conservation mandate to conserve natural diversity. A prerequisite for fulfilling this purpose is to be able to identify the species and communities that make up that biodiversity. We tested a set of varied methods for inventory and monitoring of plants, birds and terrestrial invertebrates on a grid of 40 sites in a 938 ha study area in the Slikok Creek watershed, Kenai Peninsula, Alaska. We sampled plants and lichens through observation and specimen-based methods. We surveyed birds using bird call surveys on variable circular plots. We sampled terrestrial arthropods by sweep net sampling, processing samples with High Throughput Sequencing methods. We surveyed for earthworms, using the hot mustard extraction method and identified worm specimens by morphology and DNA barcoding. We examined community membership using clustering methods and Nonmetric Multidimensional Scaling. We documented a total of 4,764 occurrences of 984 species and molecular operational taxonomic units: 87 vascular plants, 51 mosses, 12 liverworts, 111 lichens, 43 vertebrates, 663 arthropods, 9 molluscs and 8 annelid worms. Amongst these records, 102 of the arthropod species appeared to be new records for Alaska. We found three non-native species: Deroceras agreste (Linnaeus, 1758) (Stylommatophora: Agriolimacidae), Dendrobaena octaedra (Savigny, 1826) (Crassiclitellata: Lumbricidae) and Heterarthrus nemoratus (Fallén, 1808) (Hymenoptera: Tenthredinidae). Both D. octaedra and H. nemoratus were found at sites distant from obvious human disturbance. The 40 sites were grouped into five community groups: upland mixed forest, black spruce forest, open deciduous forest, shrub-sedge bog and willow. We demonstrated that, at least for a subset of species that could be detected using these methods, we were able to document current species distributions and assemblages in a way that could be efficiently repeated for the purposes of biomonitoring. While our methods could be improved and additional methods and groups could be added, our combination of techniques yielded a substantial portion of the data necessary for fulfilling Kenai National Wildlife Refuge's broad conservation purposes.

Natural resource and wildlife managers must balance the disparate priorities of a diversity of stakeholders. To manage these priorities, a firm understanding of topics salient to the public is needed. The media often report on issues of importance to the public; therefore, these reports may be a useful measure of public interest. However, efficient methods for distinguishing diverse topics related to a wildlife management issue reported in the media and changes in the salience of those topics have been lacking. We used latent Dirichlet allocation, a Bayesian mixture model, to quantitatively assess the salience of topics surrounding the gray wolf (Canis lupus), which was reintroduced to Idaho (U.S.A.) in 1995. We analyzed articles published from 1960 to 2015 in an Idaho newspaper. We identified 6 distinct topics associated with gray wolves: policy, hunting, biological status, implementation of management, recovery, and human-wolf conflict. The salience of topics pre- and postreintroduction of wolves (1995) and pre- and postdelisting of wolves from the U.S. Endangered Species Act (2009) differed significantly, underscoring that these events were turning points in how issues were being publicly discussed and framed. Articles written by the local reporters were more likely to report on topics regarding conflict between humans and wolves, whereas articles sourced from a national outlet reported more on topics pertaining to wolf policy and biological status. In the context of managing a contentious, far-ranging, and long-lived wildlife species, our methods can help guide the location and timing of a suite of management strategies (e.g., media relation plans and stakeholder engagement) that promote human-wildlife coexistence across different landscapes.Seguimiento de Medio Siglo de Reportes Mediáticos sobre Lobos Grises Resumen Los administradores de los recursos naturales y de la fauna deben balancear las prioridades discrepantes de una diversidad de accionistas. Para manejar estas prioridades se requiere de un entendimiento firme de los temas destacados para el público. Los medios frecuentemente reportan sobre temas de importancia para el público; por lo tanto, estos reportajes pueden ser una medida útil del interés del público. Sin embargo, hoy en día se carece de métodos eficientes para distinguir diferentes temas relacionados con un tema de manejo de fauna reportado en los medios y para reconocer los cambios en la prominencia de estos temas. Usamos la asignación latente Dirichlet, un modelo de mezcla bayesiana, para evaluar cuantitativamente la prominencia de los temas circundantes al lobo gris (Canis lupus), el cual fue reintroducido a Idaho (E.U.A.) en 1995. Analizamos artículos publicados en un periódico de Idaho desde 1960 y hasta 2015. Identificamos seis temas distintos asociados con el lobo gris: política, caza, estado biológico, implementación del manejo, recuperación, y conflicto humano-lobos. La prominencia de los temas previa y posterior a la introducción de los lobos (1995) y previa y posterior al retiro de los lobos del Acta de Especies Amenazadas de E.U.A (2009) varió significativamente, resaltando que estos eventos fueron puntos críticos para cómo se discutían y enmarcaban estos temas. Los artículos escritos por los reporteros locales tuvieron una mayor probabilidad de tratar temas sobre el conflicto entre humanos y lobos, mientras que los artículos surgidos en alguna fuente nacional reportaron más sobre temas correspondientes a la política y al estado biológico de los lobos. En el contexto de una especie silvestre contenciosa, de una extensión amplia y un ciclo de vida largo, nuestros métodos pueden ayudar a guiar la ubicación y el momento de un conjunto de estrategias de manejo (p. ej.: planes de relación de medios y compromiso de los accionistas) que promueva la coexistencia entre humanos y fauna a lo largo de diferentes paisajes.自然资源和野生动物管理者必须平衡各个利益相关者之间不同的优先事宜。为此, 需要明确理解公众关注的话题。媒体常常报道对公众重要的问题, 这些报道因而可以作为衡量公众兴趣的有用指标。但现在还缺乏有效的方法, 来从媒体报道的与野生动物管理有关的事件中区分出各种话题, 并分析这些话题受关注程度的变化。我们用隐狄利克雷分布 (一种贝叶斯混合模型) 定量评估了围绕灰狼 (Canis lupus) 的话题热度, 这些灰狼是在1995 年被重引入到爱达荷州 (美国) 的。我们分析了1960 -2015 年一家爱达荷州报纸上发表的文章, 确定了六个与灰狼有关的话题: 政策、狩猎、生物学地位、管理实施、恢复和人狼冲突。结果显示, 灰狼重引入 (1995年) 前后, 以及从《美国濒危物种法案》中去除 (2009年) 前后的受关注话题明显不同, 表明这两个事件是公开讨论和确定相关问题的转折点。由当地记者撰写的文章更多地报道了人狼冲突的话题, 而国家媒体发表的文章则更关注狼的相关保护政策和生物学地位。在管理那些富有争议、分布广泛和寿命长的野生动物时, 我们的方法可以帮助指导一系列管理策略 (如媒体公关计划和利益相关者的参与) 实施的位置和时机, 以促进不同的景观中人类与野生动物的共存。 【翻译: 胡怡思; 审校: 聂永刚】.

The triangular array formed from the coefficients of sin + cos is discussed in relation to Pascal's triangle. (JG)

Electrical signals are recorded from and sent into the body via the skin in a number of applications. In practice, skin is often hydrated with liquids having different conductivities so a model was produced in order to determine the relationship between skin impedance and conductivity.A model representing the skin was subjected to a variety of electrical signals. The parts of the model representing the stratum corneum were given different conductivities to represent different levels of hydration.The overall impedance and conductivity of the cells did not vary at frequencies below 40 kHz. Above 40 kHz, levels of increased conductivity caused the overall impedance to decrease.The variation in impedance with conductivity between 5 and 50 mSm

The design and fabrication of nanopores within three-dimensionally structured gold films with spherical microcavities of 1.2 µm diameter and 0.6 µm deep in hexagonal close-packed arrays, are described. The cavities are fabricated by electroplating gold around self-assembled arrays of polymer spheres. Following removal of the spheres, and 'lift-off' of the 3D structured gold film, some of the microcavities were milled with a Helium Ion Microscope to provide nanopores through the centre of the microcavity base right through the film. The geometry of the nanopore within the device is designed using theoretical approaches to provide the optimal electric field intensity in the very centre of the nanopore when excited with light of ~ 600 nm (in water). In this paper we report the theoretical simulations used to evaluate the optimal geometry of the nanopore within the centre/base of the gold microcavity. Although a number of various geometries and sizes of pores were considered the theoretical results provide evidence that a pore of 50nm with rounded corners will provide the greatest electrical field intensity inside the pore and the fabrication results provide a demonstrated practical approach for creation of these nanopores within these 3D gold structured films.

Three-dimensionally structured gold membrane films with nanopores of defined, periodic geometries are designed and fabricated to provide the spatially localised enhancement of electric fields by manipulation of the plasmons inside nanopores. Square nanopores of different size and orientation relative to the pyramid are considered for films in aqueous and air environments, which allow for control of the position of electric fields within the structure. Designs suitable for use with 780 nm light were created. Here, periodic pyramidal cavities produced by potassium hydroxide etching to the {111} planes of (100) silicon substrates are used as templates for creating a periodic, pyramidal structured, free-standing thin gold film. Consistent with the findings from the theoretical studies, a nano-sized hole of 50 nm square was milled through the gold film at a specific location in the cavity to provide electric field control which can subsequently used for enhancement of fluorescence or Raman scattering of molecules in the nanopore.

Modified triplex-forming oligonucleotides distinguish 5-methyl cytosine from unmethylated cytosine in DNA duplexes by differences in triplex melting temperatures. The discrimination is sequence-specific; dramatic differences in stabilisation are seen for CpA methylation, whereas CpG methylation is not detected. This direct detection of DNA methylation constitutes a new approach for epigenetic analysis.

An investigation of the photoinduced fluorescence enhancement (PFE) behavior of CdSe/ZnS core/shell quantum dots deposited at low densities, under anhydrous and controlled water humidity, under oxygen or argon, is presented. The photoluminescence properties of CdSe/ZnS QDs are highly dependent upon the local gaseous environment. Under anhydrous conditions, under either oxygen or argon, there was no observed PFE, even though there were remarkable differences in the photoluminescence spectra. Under argon, (i) the initial photoluminescence properties are independent of humidity level; however, (ii) the PFE effect observed is highly dependent on the environmental humidity levels. Under oxygen, (i) the initial photoluminescence properties (spectra and yield) are dependent on humidity levels and (ii) the PFE effect observed is highly dependent on the humidity levels. Comparing D2O versus H2O humidity level effects on the photoluminescence properties of CdSe/ZnS QDs provides evidence for a water-molecule-stabilized state that facilitates luminescence processes. The products of CdSe/ZnS QDs exposed under a humid oxygen environment were evaluated by X-ray photoelectron spectroscopy. Oxidation of both the CdSe core and the ZnS shell was established. Oxidation of the ZnS shell is suggested to be a result of reaction with peroxide products resulting from the oxygen radical anion. These results highlight the important sensitivity of QDs to water and prove the existence of competing electronic and chemical effects on different time scales.

By sequentially pushing micro-beads towards and away from a sensing surface, we show that ultrasonic radiation forces can be used to enhance the interaction between a functionalized glass surface and polystyrene micro-beads, and distinguish those that bind to the surface, ultimately by using an integrated optical waveguide implanted in the reflector to facilitate optical detection. The movement towards and immobilization of streptavidin coated beads onto a biotin functionalized waveguide surface is achieved by using a quarter-wavelength mode pushing beads onto the surface, while the removal of non-specifically bound beads uses a second quarter-wavelength mode which exhibits a kinetic energy maxima at the boundary between the carrier layer and fluid, drawing beads towards this surface. This has been achieved using a multi-modal acoustic device which exhibits both these quarter-wavelength resonances. Both 1-D acoustic modeling and finite element analysis has been used to design this device and investigate the spatial uniformity of the field.We demonstrate experimentally that 90% of specifically bound beads remain attached after applying ultrasound, with 80% of non-specifically bound control beads being successfully removed acoustically. This approach overcomes problems associated with lengthy sedimentation processes used for bead-based bioassays and surface (electrostatic) forces, which delay or prevent immobilization. We explain the potential of this technique in the development of DNA and protein assays in terms of detection speed and multiplexing.

"How the construction of oximeter sensors has evolved over the last decade is a reflection of the advancement in light sources and detectors from the optoelectronics and microelectronics research fields."

The fluorescence properties of thiazole orange, linked via a (1) hydrophobic alkyl or a (2) hydrophillic ethylene glycol chain to the central internucleotidic phosphate group of a pentadeca-2'-deoxyriboadenylate (dA(15)), are evaluated. Linkage at the phosphate group yields two stereoisomers, S-isomer of the phosphorus chiral center (Sp) and R-isomer of the phosphorus chiral center (Rp); these are studied separately. The character of the linkage chain and the chirality of the internucleotidic phosphate linkage site influence the fluorescent properties of these thiazole orange-oligonucleotide conjugates (TO-probes). Quantum yields of fluorescence (Phi(fl)) of between 0.04 and 0.07 were determined for the single-stranded conjugates. The fluorescence yield increased by up to five times upon hybridization with the complementary sequence (d5'[CACT(15)CAC(3')]); Phi(fl) values of between 0.06-0.35 were determined for the double-stranded conjugates. The Phi(fl) value (0.17) of thiazole orange, 1-(N,N'-trimethylaminopropyl)-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)- 2-methylidene]-quinolinium iodide (TO-Pro 1) in the presence of the oligonucleotide duplex (TO-Pro 1: dA(15)(.)d(5')[CACT(15)CAC(3')] (1:1)) is much less than that for some of the hybrids of the conjugates. Our studies, using steady-state and time-resolved fluorescence experiments, show that a number of discrete fluorescent association species between the thiazole orange and the helix are formed. Time-resolved studies on the four double-stranded TO-probes revealed that the fluorescent oligonucleotide-thiazole orange complexes are common, only the distribution of the species varies with the character of the chain and the chirality at the internucleotidic phosphate site. Those TO-probes in which the isomeric structure of the phosphate-chain linkage is Rp, and therefore such that the fluorophore is directed toward the minor groove, have higher Phi(fl) values than the Sp isomer. Of the systems studied, thiazole orange linked by an alkyl chain to the internucleotidic phosphate (Rp isomer) has the highest Phi(fl) and the greatest fraction of the longest-lived fluorescent thiazole orange species (in the hybrid form).

A new masking technology for wet etching of glass, to a depth of more than 300µm, is reported. Various mask materials, which can be patterned by standard photolithography and metal etching processes, were investigated for glass etching in concentrated hydrofluoric acid (HF). A multilayer of metal, Cr/Au/Cr/Au, in combination with thick SPR220-7 photoresist, was found to be ideal for this purpose. Through holes etched from both sides of a 500µm thick Pyrex glass wafer were obtained. Pinholes, created in the glass by failure of simple metal masking when subjected to HF etching, were successfully eliminated using the new masking technology. In addition, the lateral undercutting of glass caused by the under-etching of the Cr mask was minimized to 27% of the etching depth. With these advantages, this newly developed masking method was successfully applied to fabricate microfluidic components for a micro-peristaltic pump to be integrated in a micro polymerase chain reaction (PCR) device. These components include through holes for liquid accessing and electrical contacting and a 200µm thick pump diaphragm. This new masking technology also adds to the methods available to fabricate the microfluidic devices in glass substrates.

An investigation into the pumping flow rates and the time-resolved membrane actuation of a microperistaltic pump integrated within a micro total analysis system (µTAS) is presented. The results include (i) the design of the driver circuit to operate the peristaltic micropump, (ii) Michelson interferometer measurements of the pump displacement and (iii) pump flow rate measurements. The peristaltic micropump, configured with three PZT actuated glass membranes and silicon channels, is integrated within the µTAS device with microfluidic reaction chambers. The micropump pumps a 1 µl droplet back and forth between the reaction chambers.

Detection of DNA sequences is pivotal to many modern molecular diagnostic methods, but the ability to directly detect DNA sequences, without the need for signal amplification (such as by applying a polymerase chain reactions) is highly desirable. Here we investigate the potential for gold inverted pyramidal structures (also known as Klarite®) for DNA detection, as these simple chips containing the structured surface on a face could offer an improved format for DNA based diagnostic methods. Our strategy included optimization of the fabrication protocols to achieve flat gold surfaces within the inverted pyramidal surface and then a subsequent assessment of these substrates for direct DNA detection by Raman microscopy. These studies demonstrate for the first time the potential of these gold structured planar substrates for DNA analysis applications.

Because of the strong confinement and large refractive index contrast, silica nanowires can efficiently generate light in the UV through intermodal phase matching. Similarly, the fraction of power in the evanescent field allows for many applications in sensing.

We demonstrate an efficient method for DNA Sensing using simple devices, the microfiber coupler, and the Multi port Microcoil Resonator. These devices promise, high sensitivities, up to 200nm/RIU, and low detection limit of 10-6 RIU.

Although strategies for the immobilization of DNA oligonucleotides onto surfaces for bioanalytical and top-down bio-inspired nanobiofabrication approaches are well developed, the effect of introducing spacer molecules between the surface and the DNA oligonucleotide for the hybridization of nanoparticle–DNA conjugates has not been previously assessed in a quantitative manner. The hybridization efficiency of DNA oligonucleotides end-labelled with gold nanoparticles (1.4 or 10 nm diameter) with DNA sequences conjugated to silicon surfaces via hexaethylene glycol phosphate diester oligomer spacers (0, 1, 2, 6 oligomers) was found to be independent of spacer length. To quantify both the density of DNA strands attached to the surfaces and hybridization with the surface-attached DNA, new methodologies have been developed. Firstly, a simple approach based on fluorescence has been developed for determination of the immobilization density of DNA oligonucleotides. Secondly, an approach using mass spectrometry has been created to establish (i) the mean number of DNA oligonucleotides attached to the gold nanoparticles and (ii) the hybridization density of nanoparticle–oligonucleotide conjugates with the silicon surface–attached complementary sequence. These methods and results will be useful for application with nanosensors, the self-assembly of nanoelectronic devices and the attachment of nanoparticles to biomolecules for single-molecule biophysical studies.

We design and optimize a simple, low-cost and compact biosensor based upon a microfiber coupler, with a predicted sensitivity and detection limit up to S~104 nm/RIU and DL~10-6 RIU respectively.

Optical approaches have been explored to enable undifferentiated to be distinguished from differentiated stem cells. Optical measurements combined with theoretical and statistical analysis provide a novel approach with the potential for identification and sorting.

A novel microfluidic concentration gradient generator is designed where secondary flow, induced via a surface groove, is used to yield a concentration gradient across the output of the microfluidic device. The concentration gradient generator design consists of a single microfluidic channel with two inputs and a single obliquely angled surface groove within the base of the channel to induce the secondary flow and create the concentration gradient. The design allows a concentration gradient to be chosen, either linear or exponential, at the exit of the microfluidic channel with the shape and dimensions of the surface groove within the channel obtained by numerical optimisation. The designed device has a small footprint, suitable for integration within lab-on-a-chip structures for the delivery of a series of an agent to either (i) a single channel and with a concentration gradient or to (ii) a series of reactors with concentrations across a defined range, for bioscience or pharmaceutical screening applications or for chemical reactions. © 2012 Elsevier B.V.

Coherent anti-Stokes Raman scattering (CARS) microscopy is applied for the first time for the evaluation of the protein secondary structure of polyglutamine (polyQ) aggregates in vivo. Our approach demonstrates the potential for translating information about protein structure that has been obtained in vitro by X-ray diffraction into a microscopy technique that allows the same protein structure to be detected in vivo. For these studies, fibres of polyQ containing peptides (D2Q15K2) were assembled in vitro and examined by electron microscopy and X-ray diffraction methods; the fibril structure was shown to be cross β-sheet. The same polyQ fibres were evaluated by Raman spectroscopy and this further confirmed the β-sheet structure, but indicated that the structure is highly rigid, as indicated by the strong Amide I signal at 1659 cm-1. CARS spectra were simulated using the Raman spectrum taking into account potential non-resonant contributions, providing evidence that the Amide I signal remains strong, but slightly shifted to lower wavenumbers. Combined CARS (1657 cm-1) and multi-photon fluorescence microscopy of chimeric fusions of yellow fluorescent protein (YFP) with polyQ (Q40) expressed in the body wall muscle cells of Caenorhabditis elegans nematodes (1 day old adult hermaphrodites) revealed diffuse and foci patterns of Q40-YFP that were both fluorescent and exhibited stronger CARS (1657 cm-1) signals than in surrounding tissues at the resonance for the cross β-sheet polyQ in vitro.

Colloidal quantum dots (QDs) have received considerable attention as luminescent probes for DNA analysis applications. The underlying photophysical and photochemical properties of these probes need consideration when designing assays for DNA analysis. These properties include intermittent fluorescence often termed 'blinking', photobleaching, photoinduced fluorescence enhancement and, as a result of recent evidence, photo-induced generation of reactive oxygen species leading to DNA damage. Even though the design of assays for DNA analysis using QDs needs care, QDs do provide advantages over fluorophores for many emerging DNA analysis methods where low copy numbers of DNA are present. Many of the more traditional DNA assay methods using fluorophore labeled probes either cannot be translated, or show no benefit in using QDs as the lumophore

The analytical (numerical) design of planar microfluidic affinity chromatography devices, which consist of multiple separation lanes and multiple, different surface-immobilised receptor patterns in each lane, is described. The model is based on the analytical solution of the transport-reaction equations in microfluidic systems of low Gratz number and for injection of small analyte plugs. The results reveal a simple approach for the design of microfluidic affinity chromatography devices tailored to the separation of bioanalytes, where receptors with high binding affinity are available. These devices have been designed for bioanalytical applications in mind, most notably for the proteomics field; the results are illustrated with an example using β-Amyloid binding peptides. © 2012 Elsevier B.V.

The optical propulsion of mammalian eukaryotic cells along the surface of an integrated channel waveguide is demonstrated. 10μm diameter polymethylmethacrylate (PMMA) spherical particles and similarly sized mammalian eukaryotic cells in aqueous medium are deposited in a reservoir over a caesium ion-exchanged channel waveguide. Light from a fibre laser at 1064nm was coupled into the waveguide, causing the polymer particles or cells to be propelled along the waveguide at a velocity which is dependent upon the laser power. A theoretical model was used to predict the propulsion velocity as a function of the refractive index of the particle. The experimental results obtained for the PMMA particles and the mammalian cells show that for input powers greater than 50mW the propulsion velocity is approximately that obtained by the theoretical model. For input powers of less than ~50mW neither particles nor cells were propelled; this is considered to be a result of surface forces (which are not considered in the theoretical model). The results are discussed in light of the potential application of optical channel waveguides for bioanalytical applications, namely in the identification and sorting of mammalian cells from mixed populations without the need for fluorescence or antibody labels.

The photochemistry of substituted 1-(benzoylimino)pyridinium and 4-(N-bezoylimino)-1,2,4-triazolium ylides was investigated to judge their capacity to give aroylnitrenes and their suitability for use in photolabeling experiments. Evidence presented indicates that the triplet states of the ylides cleave to generate aroylnitrenes. In an attempt to enhance triplet formation, nitro- and acetyl-substituted pyridinium ylides were examined. Their irradiation does not give nitrenes in meaningful yield. However, irradiation of the triazolium ylides gives nitrenes in excellent yield

New designs for microfluidic channels to be integrated with small-scale affinity sensors for analytical applications are provided. Theoretical approaches demonstrate efficient and uniform mass transfer of the analyte from the bulk flow to small-scale affinity sensors in the base of fluidic channels by (i) active control of the analyte flow speed over the affinity sensor, (ii) non-rectangular channel geometries and (iii) non-uniform distributions of recognition binding sites over the active area of the sensor. The methodology reported provides generic strategies that can be exploited for small-scale sensors in single or multiplex formats. © 2007 Elsevier B.V. All rights reserved.

Injection strategies have been employed in the field of fluidic MEMS using piezo electric or thermal actuators. A very popular application for such technology is inkjet printing. Largely this technology is used to produce droplets of fluid in air; the aim of this investigation is to produce an injection device for the precise dispensing of nanolitre volumes of fluid. A novel technique for dispensing fluid using superparamagnetic beads has been investigated. The beads used (Dynal Biotech) contain a homogeneous dispersion of Fe. This is an investigation using silicon and Pyrex fabricated micro channels with smaller dimensions, such that the dimensions will be similar to those which will be used to produce a pipette device. Here results are presented using these fabricated micro channels, where the effects of using differently sized bead plugs and varying velocities are examined. The results follow our proposed theory; further analysis is required to determine the operation of a bead plug during all states of movement.

In this paper, we present both a theoretical evaluation and the fabrication of a novel polymerase chain reaction (PCR) microdevice. This microdevice contains elements for both thermal cycling and fluorescence detection. The proposed device is composed of a reaction chamber with integrated temperature sensor, heaters, p-n diode and optical filter. The advantage of combining these in a single structure is that real time detection of DNA amplification will be possible using a small volume of the PCR solution. The photodiode is covered by a thin film optical filter in order to block out the light which is used to excite the fluorophore dyes. CdS is used for the first time for such a filter and the complete microfabrication process is described.

A method is presented for the covalent attachment of oligonucleotides to silicon (100) surfaces patterned with micron-scale features. UV light exposure of hydrogen-terminated silicon (100) coated with alkenes functionalized with N-hydroxysuccinimide ester groups results in Si-C bonded monolayers. The N-hydroxysuccinimide ester surfaces act as a template for the subsequent covalent attachment of DNA oligonucleotides. In order to create patterns of surface attached DNA oligonucleotides with high density, the surface attachment chemistry has been investigated and optimised. Micron-scale patterning of surfaces was achieved by exposure with UV laser light via a mask. DNA oligonucleotide patterns, with feature sizes of several microns, were reliably produced over large areas. The patterned surfaces were characterised with scanning electron microscopy, epifluorescence microscopy and ellipsometry. Hybridisation with fluorescent label- and gold nanoparticle-conjugates of the complementary oligonucleotide is achieved. The methods offer reliable approaches for the creation of micron-scale motifs of DNA on surfaces.

The covalent attachment of DNA oligonucleotides onto crystalline silicon (100) surfaces, in patterns with submicron features, in a straightforward, two-step process is presented. UV light exposure of a hydrogen-terminated silicon (100) surface coated with alkenes functionalized with N-hydroxysuccinimide ester groups resulted in the covalent attachment of the alkene as a monolayer on the surface. Submicron-scale patterning of surfaces was achieved by illumination with an interference pattern obtained by the transmission of 248 nm excimer laser light through a phase mask. The N-hydroxysuccinimide ester surface acted as a template for the subsequent covalent attachment of aminohexyl-modified DNA-oligonucleotides. Oligonucleotide patterns, with feature sizes of 500 nm, were reliably produced over large areas. The patterned surfaces were characterized with atomic force microscopy, scanning electron microscopy, epifluorescence microscopy and ellipsometry. Complementary oligonucleotides were hybridized to the surface-attached oligonucleotides with a density of 7 x 1012 DNA oligonucleotides per square centimetre. The method will offer much potential for the creation of nano- and micro-scale DNA biosensor devices in silicon.

The design of a novel, microfluidic chip with an integrated micro peristaltic pump and chambers for DNA amplification is described. This chip contains three reaction chambers stable at 90 °C, 72 °C and 55 °C for PCR amplification, a bi-directional peristaltic pump and optical integrated detection of the droplet. A reactant droplet is to be introduced into the device, pumped back and forth between the chambers by the micro peristaltic pump for sample processing. The static behaviour of the micro pump was modelled theoretically in order to evaluate the optimal dimensions for the pump membranes and to obtain the maximum flow rate. Thermal analysis by the finite element method was performed to optimize the location of the heaters and the temperature uniformity over the three reaction chambers. Transient thermal analysis indicates that the reactant droplet can be heated/cooled in the proposed device in less than 1 s to achieve the desired temperatures.

The design and fabrication of an ordered nanoporous silicon membrane and integrated heater and temperature sensor is described. The methodology for fabrication of the nanoporous structure has been developed for integration within microelectromechanical systems. The structure is fabricated from a 500 µm thick silicon (1 0 0) wafer, which has been etched to provide 4 × 4 mm2 membranes of 50 µm thick. Quasi-ordered nanoporous silicon is created within the membrane, the nanopores are of uniform diameter (typical structures of the order of 105 ±5 nm) and smooth sidewalls to a depth of ~300 nm, in a hexagonal close-packed pattern of 200 nm nearest neighbour. The porosity of typical fabricated samples is 31.5%. On the back side of the membrane, a heater and a temperature sensor are incorporated. Three different heater/temperature sensor designs were considered theoretically and the best design was then fabricated and studied experimentally. The results obtained provide both highly ordered nanoporous silicon fabrication methodology as well as evidence that the porous membrane can be heated without deleterious effect.

Here we present the first evidence showing that eukaryotic cells can be stably trapped in a single focused Gaussian beam with an orientation that is defined by the nucleus. A mammalian eukaryotic cell (in suspension) is trapped and is re-oriented in the focus of a linearly polarized Gaussian beam with a waist of dimension smaller than the radius of the nucleus. The cell reaches a position relative to the focus that is dictated by the nucleus and nuclear components. Our studies illustrate that the force exerted by the optical tweezers at locations within the cell can be predicted theoretically; the data obtained in this way is consistent with the experimental observations.

A novel sloped interconnect for the efficient delivery of long genomic DNA fragments into a microfluidic channel is designed, fabricated and tested. Out-of-plane slopes are fabricated in silicon wafers using the deep reactive-ion etch lag phenomenon and a combination of anisotropic and isotropic etching. The final structure is capped with anodically bonded glass. Based upon a series of etch-lag calibration studies, the interconnect was designed using finite element analysis to provide a channel with flow acceleration properties appropriate to straighten DNA molecules. The efficiency of transit of the 48.5 kb DNA fragments (~16.5 µm long when fully extended) through the microfluidic device, established using quantitative real-time polymerase chain reaction, is 95 ± 7.3%

Nanosphere lithography (NSL) has been demonstrated to be a feasible, alternative technique for the fabrication of macroporous polysilicon. The diameter of the pore can be controlled by RIE of the fabricated nanospheres template using Ar/O gas mixtures at pressure of 5mTorr and rf power of 100W.

A new acoustic-optical-microfluidic system is presented for the manipulation of bead-tagged DNA molecules. Acoustic radiation forces are used to manipulate microspheres into and away from the evanescent field of a laser coupled waveguide that is integrated into the reflector of the acoustic chamber. With suitable fluorophores the presence of the target DNA can be detected with a fluorescence microscope enabling large populations of beads to be examined simultaneously. The integrated waveguide and multimodal acoustic chamber are presented here, with results showing that the microspheres can be successfully detected as they are brought into the evanescent field using a quarter-wave acoustic configuration. It is also shown that by measuring the time of flight of a microsphere between the half- and quarterwave nodal planes the bead size can be determined, providing a means of multiplexing the detection (detecting a range of different target DNA sequences).

The emission of CdSe quantum dots linked to the 5'-end of a DNA sequence is efficiently quenched by hybridisation with a complementary DNA strand with a gold nanoparticle attached at the 3'-end; contact of the quantum dot and gold nanoparticle occurs.

"Light-up" probes, icosa-alpha-thymidylate-thiazole orange conjugates, for the in situ time-resolved detection of messenger ribonucleic acid (mRNA) in living cells are evaluated. Upon annealing with polyA in aqueous solutions, the icosa-alpha-thymidylate-thiazole orange conjugates were shown to be up to 15 times more fluorescent. Microinjection of these probes into adherent fibroblasts resulted in high yields of hybridization and fluorescent signals. Incubation of cells in the presence of these probes resulted in facile internalization of the probe and similar painting of the messenger RNA in the nuclear and cytosolic regions.

Full details and a step-by-step guide suitable for printing proteins aligned to micron-sized sensors and subsequent integration and alignment of microfluidic structures are presented. The precise alignment and grafting of micron-sized biomolecule patterns with an underlying substrate at predefined locations is achieved using a novel semi-automated microcontact printer. Through integration of optical alignment methods in the x, y, and z directions, uniform contact of micron-sized stamps is achieved. Feature compression of the stamp is avoided by fine control of the stamp during contact. This printing method has been developed in combination with robust, compatible bioconjugate chemistry for patterning of a dextran-functionalized silicon oxide substrate with a NeutrAvidin-“inked” stamp and subsequent incubation with a biotin-functionalized protein. The bioconjugate chemistry is such that uniform coverage of the protein (without denaturation) over the printed motif is obtained and reproduction of the initial mask shape and dimensions is achieved. Later integration with a microfluidic structure aligned with the printed motif on the substrate is also described.

The stochastic motion behaviour of teratocarcinoma cells on PEG functionalised surfaces is investigated and analysed. The solution of 1 x 10 6 cells per ml concentration is pipetted into a reservoir and images are captured and analysed using an in-house written software. A theoretical model was used to predict the motion and compared to the experimental results. The conditions and limitations to allow teratocarcinoma cells (naturally adherent cells) to move freely in stochastic motion on surface are discussed in this paper. PEG functionalisation of the glass surface was found to improve the cells mobility, on average 26%. Analysis technique proposed in this paper demonstrates that size distribution of different cell lines can be determined. The results are presented in light of the potential application of the observed motion on functionalised surfaces for lab-on-a-chip devices, especially for adherent biological cells applications.

Realisation of a device intended for the manipulation and detection of bead-tagged DNA and other bio-molecules is presented. Acoustic radiation forces are used to manipulate polystyrene micro-beads into an optical evanescent field generated by a laser pumped ion-exchanged waveguide. The evanescent field only excites fluorophores brought within approximately 100 nm of the waveguide, allowing the system to differentiate between targets bound to the beads and those unbound and still held in suspension. The radiation forces are generated in a standing-wave chamber that supports multiple acoustic modes, permitting particles to be both attracted to the waveguide surface and also repelled. To provide further control over particle position, a novel method of switching rapidly between different acoustic modes is demonstrated, through which particles are manipulated into an arbitrary position within the chamber. A novel type of assay is presented: a mixture of streptavidin coated and control beads are driven towards a biotin functionalised surface, then a repulsive force is applied, making it possible to determine which beads became bound to the surface. It is shown that the quarter-wave mode can enhance bead to surface interaction, overcoming potential barriers caused by surface charges. It is demonstrated that by measuring the time of flight of a microsphere across the device the bead size can be determined, providing a means of multiplexing the detection, potentially detecting a range of different target molecules, or varying bead mass.